The general objective of the proposed research is to elucidate the microscopic changes which occur at the active sites of enzymes which effectively alter the catalytic properties. The work will center on studies with the glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GPD), isolated from yeast and from the bacteria B. stearothermophilus. The investigation will involve some "classical" physical organic techniques (e.g., structure-reactivity relationships) and inhibition studies. Reversible and irreversible inhibitors which are analogs of reactive intermediates (e.g., "transition state analogs") will be used to probe the effects of substituents and effectors on the individual steps in the reaction. Inhibitors which mimic the transition state of the catalytic process are useful in distinguishing between mechanistic alternatives and as a basis for drug design (i.e., potent enzyme-specific antimetabolites). We will examine the role of specific interactions between the acyl substituent and the enzyme on individual steps in the overall oxidative-phosphorylation reaction. We will also examine the catalytic role of NAD in the phosphorolytic deacylation of the acyl-enzyme (How does NAD accelerate the reaction and induce the specificity of this step? How do modulations in subunit-subunit interactions, mediated by NAD binding, affect this step?) Since the effects of NAD and enzyme-substrate interactions involve conformation changes in proteins which can be analyzed by a variety of probes, GPD can effectively serve as a model for many of the dynamic processes in biology which are mediated and controlled through conformation changes in proteins.